Early in 2003, Larry Zwiebel, Ph.D., stepped out of a cab in front of a non-descript office building on the Seattle waterfront. It was the unlikely looking headquarters for an organization with billions of dollars at its disposal, but it was the address in the invitation he had received from the Bill & Melinda Gates Foundation.

Zwiebel, professor of Biological Sciences at Vanderbilt University, was among 30 “vector biologists” summoned to Seattle to advise members of a blue-ribbon scientific panel on a major new initiative they were planning.

Vector biologists study insects and other agents that spread infectious diseases. Zwiebel’s “vector” is the Anopheles gambiae mosquito, which transmits malaria, and his goal is to understand the insect’s olfactory system at the molecular level.

Six months earlier, Bill and Melinda Gates had decided to use the extraordinary resources of their foundation—some $27 billion—to make a major impact on global health, and to do so in an extraordinarily aggressive manner.

A major component of this effort would be a “Grand Challenges in Global Health” initiative to catalyze scientific breakthroughs against the diseases that kill millions of people each year in the world’s poorest countries.

The initiative had been launched with a $200 million Gates grant to the Foundation for the National Institutes of Health (NIH), which fosters public-private partnerships to improve health through scientific discovery.

To ensure it got the biggest bang for its megabucks, a 22-member board of distinguished scientists chaired by Nobel laureate and former NIH Director Harold Varmus, M.D., had asked top experts in a number of related fields to come to Seattle for a brainstorming session.

“The atmosphere was almost somber,” Zwiebel recalls. “They made it clear that this was a very important challenge, one that they took very seriously.”

During the three-day meeting, Zwiebel and his colleagues were divided into working groups. Each group was asked to come up with a consensus set of suggestions. Board members apparently liked what his group had produced because many of its suggestions were incorporated into the final 14 “challenges” announced in October 2003.

The object was not simply to discover new knowledge, but also to create “deliverable technologies”—health tools that are both effective and practical. The tools should be inexpensive to produce, easy to distribute and simple enough for people in developing countries to use.

This approach struck a strong cord with Zwiebel. “Ever since… graduate school, I have wanted an opportunity to make a difference,” he says. “I have nothing but love and admiration for basic science, but the idea of working toward a practical application is very satisfying.”

So Zwiebel began rounding up his “dream team” of insect olfactory researchers to take advantage of this opportunity.

First, there was John Carlson, Ph.D., of Yale, an ongoing collaborator who had developed a method for transplanting the mosquito olfactory system into the intensively studied fruit fly Drosophila melanogaster.

Next was Willem Takken, Ph.D., at Wageningen University in the Netherlands, who Zwiebel got to know while he was on a post-doctoral fellowship in Heidelberg.

Takken, who is a leading expert on mosquito behavior, had extensive experience and contacts in Africa. He was instrumental in recruiting Gerry Killeen, Ph.D., from the Ifakara Health Research and Development Centre in Tanzania and David Conway, Ph.D., from the Medical Research Council Laboratories in The Gambia.

Deadliest animal

“Every one of the people who are involved, both the principals and the supporting scientists, have made critical insights and contributions over the last two decades. So, for me, it is really an honor to act as the coordinator,” Zwiebel says.

The Seattle meeting was the beginning of what proved to be an extremely long and arduous process. At the end of 2003, the scientific board issued a request for letters of intent from research groups interested in participating, and received more than 1,500.

After reviewing these letters, the board invited 450 groups, including the Vanderbilt-led team, to submit proposals.

“This was the most extensive grant application I have ever been involved with,” Zwiebel says. It took him and his colleagues several hectic months filled with international teleconference calls and thousands of e-mails to prepare their proposal.

“We were all exhausted when we finally submitted our proposal in June 2004,” he recalls. “I felt completely drained but, at the same time, I felt that it was the best proposal that I had ever written and that we said exactly what we wanted to say.”

Six month later, Zwiebel and his colleagues learned that their effort had been successful: Theirs was one of only 40-odd proposals selected for negotiations for funding. The negotiations continued for several months before the initiative announced, in June 2005, that it had offered 43 grants totaling $436.6 million to support research into a broad range of innovative research projects.

The bulk of the money—$450 million—was provided by the Gates Foundation, with an additional $27.1 million coming from the Wellcome Trust of the United Kingdom and $4.5 million from the Canadian Institutes of Health Research. The three organizations jointly administer the initiative along with the Foundation for the NIH.

The grants fell into several broad areas: developing improved vaccines to prevent diseases like malaria, tuberculosis and HIV; discovering ways to prevent the development of drug resistance; growing more nutritious crops to combat malnutrition; developing better methods for diagnosing and tracking disease in poor countries; and developing new ways to prevent insects from transmitting diseases.

Zwiebel’s group received an initial grant of $8.5 million for five years to develop ways to disrupt malaria transmission by chemical manipulation of the malaria mosquito’s sense of smell.

Theirs was one of seven grants that targeted the mosquito, reflecting the fact that the tiny insect is considered to be the deadliest animal on the planet. Malaria kills somewhere between 700,000 to 2.7 million people every year; two other mosquito-transmitted diseases, dengue fever and yellow fever, kill another 630,000. The World Health Organization estimates that a child dies of one of these diseases every 30 seconds.

“I was very impressed with the seriousness of the effort and the way they are trying to be circumspect in addressing what clearly are very complex issues,” says Zwiebel.

“Malaria control is not going to be a unilateral process,” he says. “We see vector control as one component in an overall multi-lateral strategy with more than one arrow… bed nets, vaccine development, vector control, water treatment, housing improvement—these are the elements that are going to bring malaria into a controllable strategy.”

The arrow in the Zwiebel group’s quiver is applying knowledge about how the mosquito’s “nose” works in order to identify chemical compounds that act as “super-repellants” and “super-attractants.”

The mosquito’s “nose” is located in its antennae and other head appendages. Studies at Vanderbilt and elsewhere have shown that its olfactory system consists of an array of different odorant receptors, each of which responds to a very narrow range of chemical signals.

These findings suggest that it should be possible to identify the specific human odorants and their respective receptors that allow female mosquitoes to identify their hosts when they need blood to reproduce. They also raise the possibility of identifying other chemicals that interact with these receptors in combinations that either attract or repel these highly selective insects.

Super-repellants

Previous studies have shown that human sweat contains about 350 different aromatic compounds, but not much research has been done on them, so researchers do not know much about individual variations in these odorants. Recent evidence indicates that mosquitoes use a blend of many odorants in targeting prey. So the researchers realize that they are faced with deciphering a highly complex system.

To meet the challenge, Zwiebel and his colleagues are setting up an international research network that extends from Vanderbilt, Yale and the Netherlands to Tanzania and The Gambia. It is designed to identify odorants and the receptors that mediate attraction or repulsion to humans for An. gambiae, and then to use this information to try to reduce the transmission of malaria in areas where the disease is endemic.

The network begins in the high-tech genetic engineering and molecular biology laboratories at Vanderbilt and Yale, to identify chemical compounds that interact strongly with receptors in the female mosquito’s antennae, and which appear to be related to host selection.

The mosquito’s olfactory receptors are members of a diverse family of proteins called G protein-coupled receptors (GPCRs) that are embedded in the membranes of nearly every cell, and which are the most common conduit for signaling pathways found in nature.

Two thirds of all drugs used in humans target GPCRs. Pharmaceutical companies and academic medical centers like Vanderbilt have developed large-scale, high-throughput screens as part of their drug discovery programs to find small molecules that interact with them.

The Zwiebel group has joined forces with the drug discovery team at the Vanderbilt Institute of Chemical Biology to identify novel sets of odorants that stimulate the mosquito receptors.

These compounds will be tested in the Carlson lab at Yale for their ability to activate olfactory receptors that have been transplanted into the fruit fly, the “lab rat” of genetic research as well as in the Zwiebel lab at Vanderbilt where they will be expressed in eggs of the frog genus Xenopus, and in defined cell-culture systems.

Because Drosophila has been so extensively studied for years, it provides a platform for studying mosquito receptors that is much easier than working with Anopheles. Xenopus and cell culture systems provide addition options as well as the ability to carry out high throughput screens for active molecules.

The most interesting molecules, which the researchers have dubbed BDOCs (Behaviorally Disruptive Olfactory Compounds), will be shipped to Wageningen University, where their effects on the physiology and behavior of individual mosquitoes will be analyzed. This information will be sent back to Vanderbilt and Yale to provide additional guidance in their search for candidate compounds using medicinal chemistry approaches.

BDOCs that pass the behavioral tests will be forwarded to Tanzania, where the researchers will combine them into different blends and evaluate how they affect laboratory-reared mosquitoes in a large biosphere that simulates the natural environment. They will also explore practical methods for producing these compounds from naturally occurring sources.

Finally, the most promising blends will be field tested in cooperating villages near Ifakara and in The Gambia by members of the research team. Compounds that are effective “super-repellants” could be embedded in mosquito netting to keep mosquitoes from finding their prey, while “super-attractants” could be placed in pesticide-laden traps.

The project’s goal is to develop the first target BDOCs in late 2006, and deliver them for testing in Tanzania the next year.

Zwiebel and his colleagues decided to work on chemical interventions, as opposed to the genetic approaches proposed by some of their colleagues, in part because they feel that chemical methods will be adopted more readily by the people who live in the areas where malaria is endemic.

“There is a long history of chemical intervention and insecticides,” Zwiebel says. “People have been slapping everything from mud to animal feces all over themselves for thousands of years to protect themselves from insect attacks.

“We are essentially bringing 21st century technology to a very well-established set of paradigms. I don’t think we have to convince many people that developing new, safe, economically effective repellants is a good idea.”

Surgical strike

Other Grand Challenges projects are pursuing genetic strategies to interfere with the ability of mosquitoes and other insects to transmit diseases. There are concerns, however, that development and release of transgenic insects may have unpredictable and undesirable ecological consequences.

In comparison, the Vanderbilt-led team is trying to target the mosquito’s sense of smell with compounds that will have a minimal impact on the environment. “We don’t have to worry about making an environmental faux pas with regard to releasing something that is never coming back,” Zwiebel comments.

The potential of an olfactory strategy has been demonstrated by the use of scented baits to kill the African tsetse fly. The program, which has replaced the practice of treating large tracts of land with persistent insecticides, is widely considered to be an environmental and technological success.

“One of the concerns that we have from an ecological point of view is to only target the disease vector mosquitoes that we want to repel,” says Zwiebel. “Many insects play critical roles in ecosystems in agricultural and other contexts that we do not want to affect unnecessarily. This can be potentially very devastating.”

Instead, the researchers are focusing on the receptors they have characterized that are considerably different from those found in other insects. They figure that this gives them the opportunity to develop a “surgical strike capacity” that will target the Anopheles mosquito and leave all of the other insects alone.

“We are not talking about eradicating anything and we are not talking about species replacement. We want to take as ecologically benign an approach as possible in order to reduce the contact between humans and Anopheles mosquitoes. That is where our interest ends,” Zwiebel says.

Fortunately, he and his colleagues have plenty to work with. They have characterized 77 novel odorant receptors as potential targets. Not only does this increase the odds that they can find odorant blends which are highly selective, it also gives them an opportunity to eliminate the problem of resistance.

It hasn’t taken mosquitoes long to develop resistance to many insecticides. That is because mutations in one or two genes are enough to make the chemicals ineffective.

The same would hold true for a repellant that acted on a single receptor. However, if the scientists can produce repellants containing a blend of compounds that stimulate a number of receptors, it is virtually impossible for the insects to develop resistance to them, Zwiebel says.

Although the Grand Challenge is focused on a practical outcome, Zwiebel expects it to yield new information about the molecular basis of behavior that could apply not only to insects but to other animals, including humans.

Researchers are now in a good position to understand peripheral elements of the olfactory system, such as how the mosquito senses the chemical environment at the extreme end of its nervous system, and how that chemical information—in mosquitoes and other insects—is translated into neuronal information.

“At the other end, we are also focused on the behavioral output,” he continues. “What can we do to change an attractive behavior into a repulsive behavior? Is it a concentration effect? Is it a level of stimulation of the neurons?” These are some of the basic questions that the researchers hope to answer in the course of the project.

“The fact that we can use these tools to understand fundamental neuroscience questions while, at the same time, making an important contribution to global health issues… It’s almost too good to be true,” Zwiebel says.